Expression of the human retinoblastoma gene product pp110RB in insect cells using the baculovirus system.
The product of the retinoblastoma susceptibility gene (RB) was overproduced in cultured insect cells using the baculovirus expression system. Upon insertion of the cloned human RB complementary DNA sequence into the viral genome downstream of the promoter of the polyhedrin gene, full-length RB protein with an apparent molecular weight of 110,000 was expressed in the insect cells. This protein was found to be phosphorylated, located in the nuclei of the infected cells, and immunologically indistinguishable from pp110RB of human cells as assayed by several anti-RB antibodies. Following cell disruption and a one-step immunoaffinity chromatographic purification, 6-12 mg of soluble pp110RB with approximately 95% purity were obtained per liter of infected suspension culture. Characterization of the two known biochemical properties of RB protein showed that this purified protein from insect cells behaved similarly to the authentic human pp110RB. First, it bound to DNA, and second, it could form a specific complex with SV40 T antigen in vitro. Prompt translocation of the protein from cytoplasm to nucleus after microinjection further indicated that the purified RB protein may be active. The availability of soluble, intact, and presumably active pp110RB in large quantity represents a significant advance for studying the biochemical and biophysical properties of the RB gene product as well as its potential biological function in cancer suppression.
Wang
N P
NP
Department of Pathology, University of California, San Diego, La Jolla 92093.
Qian
Y W
YW
Chung
A E
AE
Lee
W H
WH
Lee
E Y
EY
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009036:Moths; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000235:genetics*
D000952:Antigens, Polyomavirus Transforming; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D016160:Retinoblastoma Protein